Multiple-Locus Variable Number Tandem Repeat Analysis for Streptococcus pneumoniae: Comparison with PFGE and MLST

In the era of pneumococcal conjugate vaccines, surveillance of pneumococcal disease and carriage remains of utmost importance as important changes may occur in the population. To monitor these alterations reliable genotyping methods are required for large-scale applications. We introduced a high throughput multiple-locus variable number tandem repeat analysis (MLVA) and compared this method with pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The MLVA described here is based on 8 BOX loci that are amplified in two multiplex PCRs. The labeled PCR products are sized on an automated DNA sequencer to accurately determine the number of tandem repeats. The composite of the number of repeats of the BOX loci makes up a numerical profile that is used for identification and clustering. In this study, MLVA was performed on 263 carriage isolates that were previously characterized by MLST and PFGE. MLVA, MLST and PFGE (cut-off of 80%) yielded 164, 120, and 87 types, respectively. The three typing methods had Simpson's diversity indices of 98.5% or higher. Congruence between MLST and MLVA was high. The Wallace of MLVA to MLST was 0.874, meaning that if two strains had the same MLVA type they had an 88% chance of having the same MLST type. Furthermore, the Wallace of MLVA to clonal complex of MLST was even higher: 99.5%. For some isolates belonging to a single MLST clonal complex although displaying different serotypes, MLVA was more discriminatory, generating groups according to serotype or serogroup. Overall, MLVA is a promising genotyping method that is easy to perform and a relatively cheap alternative to PFGE and MLST. In the companion paper published simultaneously in this issue we applied the MLVA to assess the pneumococcal population structure of isolates causing invasive disease in the Netherlands before the introduction of the 7-valent conjugate vaccine

Molecular Identification of Bloodmeal Source in Ixodes ricinus Ticks Using 12S rDNA As a Genetic Marker

We developed an efficient molecular method for the identification of the bloodmeal sources in the tick Ixodes ricinus (L.), the European vector of the agents of Lyme borreliosis and tick-borne encephalitis. A ≈145-bp orthologous fragment of the vertebrate mitochondrial 12S rDNA was used as a molecular marker to discriminate host vertebrate species. The method consists of a single run polymerase chain reaction amplification of the 12S rDNA molecular marker by using nondegenerate primers followed by a reverse line blot hybridization assay by using specific oligonucleotide probes. The palette of probes allowed us to distinguish major groups of host vertebrates (e.g., mammals, small rodents, artiodactyls, birds, lizards) and to identify the bloodmeal sources at the genus or species level. External primers were designed and used to sequence the 12S rDNA molecular marker of a broad range of known or potential host vertebrate species (n = 60), including mammal (n = 28), bird (n = 31), and reptile (n = 1) species. The use of this technique coupled with known methods for identification of tick-borne pathogens (e.g., Borrelia burgdorferi sensu lato) allowed us to determine the source of infective bloodmeal and to identify reservoir species. The present method was successfully used to identify the source of bloodmeals in all feeding I. ricinus ticks and in half of questing field-collected I. ricinus ticks. Moreover, the bloodmeal source was identified in 65% of ticks infected with B. burgdorferi sensu lato. Further development of this technique may be envisaged for the detection of other vector-borne pathogens and their reservoir host

Bordetella pertussis, the Causative Agent of Whooping Cough, Evolved from a Distinct, Human-Associated Lineage of B. bronchiseptica

Bordetella pertussis, B. bronchiseptica, B. parapertussis(hu), and B. parapertussis(ov) are closely related respiratory pathogens that infect mammalian species. B. pertussis and B. parapertussis(hu) are exclusively human pathogens and cause whooping cough, or pertussis, a disease that has resurged despite vaccination. Although it most often infects animals, infrequently B. bronchiseptica is isolated from humans, and these infections are thought to be zoonotic. B. pertussis and B. parapertussis(hu) are assumed to have evolved from a B. bronchiseptica–like ancestor independently. To determine the phylogenetic relationships among these species, housekeeping and virulence genes were sequenced, comparative genomic hybridizations were performed using DNA microarrays, and the distribution of insertion sequence elements was determined, using a collection of 132 strains. This multifaceted approach distinguished four complexes, representing B. pertussis, B. parapertussis(hu), and two distinct B. bronchiseptica subpopulations, designated complexes I and IV. Of the two B. bronchiseptica complexes, complex IV was more closely related to B. pertussis. Of interest, while only 32% of the complex I strains were isolated from humans, 80% of the complex IV strains were human isolates. Comparative genomic hybridization analysis identified the absence of the pertussis toxin locus and dermonecrotic toxin gene, as well as a polymorphic lipopolysaccharide biosynthesis locus, as associated with adaptation of complex IV strains to the human host. Lipopolysaccharide structural diversity among these strains was confirmed by gel electrophoresis. Thus, complex IV strains may comprise a human-associated lineage of B. bronchiseptica from which B. pertussis evolved. These findings will facilitate the study of pathogen host-adaptation. Our results shed light on the origins of the disease pertussis and suggest that the association of B. pertussis with humans may be more ancient than previously assumed

PFGE diversity within the methicillin-resistant Staphylococcus aureus clonal lineage ST398

<p>Abstract</p> <p>Background</p> <p>Livestock has recently been identified as a new reservoir of methicillin-resistant <it>Staphylococcus aureus </it>(MRSA). Most isolates belong to ST398 and are non-typeable with PFGE using <it>Sma</it>I, making it difficult to study transmission and outbreaks. Therefore, a new PFGE using <it>Cfr</it>9I, a neoschizomer of <it>Sma</it>I was optimized and evaluated to investigate ST398 isolates.</p> <p>Results</p> <p>After optimizing and evaluating the <it>Cfr</it>9I PFGE, clear and reproducible banding patterns were obtained from all previously non-typeable MRSA (NT_{<it>Sma</it>I}-MRSA) isolates. The PFGE patterns of ST398 isolates showed more diversity than with <it>spa</it>-typing and/or MLST. The PFGE results showed diversity within and between the two most prevalent <it>spa</it>-types of NT_{<it>Sma</it>I}-MRSA (t011 and t108). No match was found, when comparing banding patterns of the NT_{<it>Sma</it>I}-MRSA with 700 different PFGE types, obtained with <it>Sma</it>I digestion, in our database of more than 4000 strains. Furthermore, possible transmission among veterinarians and their family members was investigated and an outbreak of ST398 MRSA in a residential care facility was confirmed with the <it>Cfr</it>9I PFGE.</p> <p>Conclusions</p> <p>The adjusted PFGE can be used as a method for selecting important and distinct ST398 isolates for further research. The adjustments in the PFGE protocol using <it>Cfr</it>9I are easy to implement to study the ST398 clonal lineage in laboratories which already have a PFGE facility.</p

Population Structure of Invasive Streptococcus pneumoniae in the Netherlands in the Pre-Vaccination Era Assessed by MLVA and Capsular Sequence Typing

The introduction of nationwide pneumococcal vaccination may lead to serotype replacement and the emergence of new variants that have expanded their genetic repertoire through recombination. To monitor alterations in the pneumococcal population structure, we have developed and utilized Capsular Sequence Typing (CST) in addition to Multiple-Locus Variable number tandem repeat Analysis (MLVA)

Molecular characterization of MRSA collected during national surveillance between 2008 and 2019 in the Netherlands

BACKGROUND: Although the Netherlands is a country with a low endemic level, methicillin-resistant Staphylococcus aureus (MRSA) poses a significant health care problem. Therefore, high coverage national MRSA surveillance has been in place since 1989. To monitor possible changes in the type-distribution and emergence of resistance and virulence, MRSA isolates are molecularly characterized.METHODS: All 43,321 isolates from 36,520 persons, collected 2008-2019, were typed by multiple-locus variable number tandem repeats analysis (MLVA) with simultaneous PCR detection of the mecA, mecC and lukF-PV genes, indicative for PVL. Next-generation sequencing data of 4991 isolates from 4798 persons were used for whole genome multi-locus sequence typing (wgMLST) and identification of resistance and virulence genes.RESULTS: We show temporal change in the molecular characteristics of the MRSA population with the proportion of PVL-positive isolates increasing from 15% in 2008-2010 to 25% in 2017-2019. In livestock-associated MRSA obtained from humans, PVL-positivity increases to 6% in 2017-2019 with isolates predominantly from regions with few pig farms. wgMLST reveals the presence of 35 genogroups with distinct resistance, virulence gene profiles and specimen origin. Typing shows prolonged persistent MRSA carriage with a mean carriage period of 407 days. There is a clear spatial and a weak temporal relationship between isolates that clustered in wgMLST, indicative for regional spread of MRSA strains.CONCLUSIONS: Using molecular characterization, this exceptionally large study shows genomic changes in the MRSA population at the national level. It reveals waxing and waning of types and genogroups and an increasing proportion of PVL-positive MRSA.</p

Vaccine Preventability of Meningococcal Clone, Greater Aachen Region, Germany

An emerging serogroup B clone can be prevented by vaccines

Genomic comparison of mecC-carrying methicillin-resistant Staphylococcus aureus from hedgehogs and humans in the Netherlands

Objectives: MRSA carrying the mecC gene (mecC-MRSA) have been found in humans and animals worldwide. A high carriage rate of mecC-MRSA has been described among hedgehogs in different countries. We performed genomic comparison of mecC-MRSA from hedgehogs and humans using next-generation sequencing (NGS) to investigate possible zoonotic transmission in the Netherlands. Methods: Nasal swabs from hedgehogs (n = 105) were cultured using pre-enrichment and selective plates. Isolates were sequenced using Illumina NGS platforms. These data were compared with sequence data of mecC-MRSA (n = 62) from the Dutch national MRSA surveillance in humans. Results: Fifty hedgehogs were found to be MRSA positive, of which 48 carried mecC. A total of 60 mecC-MRSA isolates derived from 50 hedgehogs were compared with the human isolates. Fifty-nine mecC-MRSA from hedgehogs and all but one isolate from humans belonged to clonal complexes CC130 and CC1943. The mecC gene was located within the SCCmec XI element. Most mecC-MRSA did not carry other resistance genes besides mecC and blaZ. Two human isolates carried erm(C). Isolates differed in the presence of various virulence genes, which were linked to distinct STs and clonal complexes. Some isolates had up to 17 virulence genes, which underlines their pathogenic potential. No genetic clusters of hedgehog and human isolates were found. Conclusions: mecC-MRSA from hedgehogs and humans mainly belonged to the same two clonal complexes, indicating a common source. No firm evidence for recent zoonotic transmission was found. Further studies are needed to investigate the role of hedgehogs in the occurrence of mecC-MRSA in humans

Livestock-associated methicillin-resistant Staphylococcus aureus epidemiology, genetic diversity, and clinical characteristics in an urban region

ObjectivesWhile Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA), defined as CC398, is a well-known pathogen among those working with livestock, there are indications that LA-MRSA prevalence among the general population is increasing. However, the clinical impact in urban areas remains unknown. The aim of this study was to assess the genetic epidemiology and clinical characteristics of LA-MRSA in an urban area with a limited livestock population.MethodsIn this retrospective study, we evaluated LA-MRSA strains that were collected between 2014 and 2018 from patients who received clinical care in a single urban area in Netherlands. Patient files were assessed for livestock exposure data, clinical findings, and contact tracing information. Next-generation sequencing (NGS) analysis in combination with wgMLST was conducted to assess genetic diversity and relatedness and to detect virulence and resistance genes.ResultsLA-MRSA strains were cultured from 81 patients, comprising 12% of all the MRSA strains found in seven study laboratories between 2014 and 2018. No livestock link was found in 76% of patients (n = 61), and 28% of patients (n = 23) had an infection, mostly of the skin or soft tissue. Contact tracing had been initiated in 14 cases, leading to the identification of two hospital transmissions: a cluster of 9 cases and one of 2 cases. NGS data were available for 91% (n = 75) of the patients. wgMLST confirmed the clusters detected via contact tracing (n = 2) and identified 5 additional clusters without a known epidemiological link. Relevant resistance and virulence findings included the PVL virulence gene (3 isolates) and tetracycline resistance (79 isolates).ConclusionLA-MRSA may cause a relevant burden of disease in urban areas. Surprisingly, most infections in the present study occurred in the absence of a livestock link, suggesting inter-human transmission. These findings and the presence of PVL and other immune evasive complex virulence genes warrant future surveillance and preventative measures